ABSTRACT
The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of Chlamydomonas reinhardtii that we captured by a cyclic guanosine monophosphate (cGMP) affinity chromatography approach, namely, soluble starch synthase STA3, starch-branching enzyme SBE1, and α-amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzyme's N-terminal aspartate kinase-chorismate mutase-tyrA domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N and C metabolism at the enzymatic level, increasing the liberation of C skeletons from starch when high Gln levels signal an abundance of assimilated N.
ABSTRACT
Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.
Subject(s)
Amidohydrolases , Anti-Bacterial Agents , Escherichia coli , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/metabolism , Amidohydrolases/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Escherichia coli/enzymology , Escherichia coli/drug effects , Crystallography, X-Ray , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Models, Molecular , Humans , Structure-Activity RelationshipABSTRACT
[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.
Subject(s)
Aquaporins , Hydrogenase , Iron-Sulfur Proteins , Hydrogen/chemistry , Hydrogenase/chemistry , Protons , Oxygen/chemistry , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolismABSTRACT
The lack of new antibiotics and the rapidly rising number of pathogens resistant to antibiotics pose a serious problem to mankind. In bacteria, the cell membrane provides the first line of defence to antibiotics by preventing them from reaching their molecular target. To overcome this entrance barrier, it has been suggested[1] that small Gold-Nanoparticles (AuNP) could possibly function as drug delivery systems for antibiotic ligands. Using actinonin-based ligands, we provide here proof-of-principle of AuNP functionalisation, the capability to bind and inhibit the target protein of the ligand, and the possibility to selectively release the antimicrobial payload. To this end, we successfully synthesised AuNP coated with thio-functionalised actinonin and a derivative. Interactions between 15N-enriched His-peptide deformylase 1-147 from E. coli (His-ecPDF 1-147) and compound-coated AuNP were investigated via 2D 1H-15N-HSQC NMR spectra proving the direct binding to His-ecPDF 1-147. More importantly by adding dithiothreitol (DTT), we show that the derivative is successfully released from AuNPs while still bound to His-ecPDF 1-147. Our findings indicate that AuNP-conjugated ligands can address and bind intracellular target proteins. The system introduced here presents a new delivery platform for antibiotics and allows for the easy optimisation of ligand coated AuNPs.
Subject(s)
Amidohydrolases , Gold , Metal Nanoparticles , Gold/chemistry , Escherichia coli , Ligands , Metal Nanoparticles/chemistry , Anti-Bacterial Agents/pharmacology , Hydroxamic AcidsABSTRACT
[FeFe]-hydrogenases are efficient H2 converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover. Kinetic assays via protein film electrochemistry show the CpI variant C299D is significantly less inhibited by HCHO, corroborating the structural results. By combining our data from protein crystallography, site-directed mutagenesis and protein film electrochemistry, a reaction mechanism involving the cofactor's amine base, the thiol group of C299 and HCHO can be deduced. In addition to the specific case of [FeFe]-hydrogenases, our study provides additional insights into the reactions between HCHO and protein molecules.
Subject(s)
Hydrogenase , Iron-Sulfur Proteins , Hydrogenase/chemistry , Protons , Catalysis , Formaldehyde/pharmacology , Amines , Hydrogen/chemistry , Iron-Sulfur Proteins/chemistryABSTRACT
Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria, and fungi. Land plant phytochromes use phytochromobilin (PΦB) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IXα (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to PФB by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only PФB biosynthesis-related genes (HY2). The HY2 of the streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) has already indirectly been indicated to participate in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in Escherichia coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we confirmed the product and identified intermediates of the reaction. Site-directed mutagenesis revealed 2 aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a PΦB-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of 2 additional members of the HY2 lineage enabled us to define 2 distinct clades, the PCB-HY2 and the PΦB-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.
Subject(s)
Cyanobacteria , Phytochrome , Phylogeny , Ferredoxins/genetics , Plants/metabolism , Bile Pigments/metabolism , Biliverdine/chemistry , Biliverdine/genetics , Biliverdine/metabolism , Phytochrome/genetics , Phytochrome/metabolism , Cyanobacteria/genetics , Cyanobacteria/metabolismABSTRACT
The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288â¯K of k 1 = 0.49 ± 0.28â¯s-1 and k 2 = 0.014 ± 0.003â¯s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4â¯s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.
Subject(s)
Bacterial Proteins , Escherichia coli Proteins , Bacterial Proteins/metabolism , Spectroscopy, Fourier Transform Infrared , Hydrolysis , Adenosine Triphosphate/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolismABSTRACT
Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN- ) ligands. Extrinsic CO and CN- , however, inhibit hydrogenases. The mechanism by which CN- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39â Å allowed us to distinguish intrinsic CN- and CO ligands and to show that extrinsic CN- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.
Subject(s)
Hydrogenase , Iron-Sulfur Proteins , Protons , Hydrogen/chemistry , Hydrogenase/metabolism , Cyanides/metabolism , Catalysis , Iron-Sulfur Proteins/chemistryABSTRACT
AIMS: Actinobacteria are known to produce extracellular enzymes including DyPs. We set out to identify and characterize novel peroxidases from Streptomyces chartreusis NRRL 3882, because S. chartreusis belongs to the small group of actinobacteria with three different DyPs. METHODS AND RESULTS: The genome of the actinomycete S. chartreusis NRRL 3882 was mined for novel DyP-type peroxidases. Three genes encoding for DyP-type peroxidases were cloned and overexpressed in Escherichia coli. Subsequent characterization of the recombinant proteins included examination of operating conditions such as pH, temperature and H2 O2 concentrations, as well as substrate spectrum. Despite their high sequence similarity, the enzymes named SCDYP1-SCDYP3 presented distinct preferences regarding their operating conditions. They showed great divergence in H2 O2 tolerance and stability, with SCDYP2 being most active at concentrations above 50 mmol l-1 . Moreover, SCDYP1 and SCDYP3 preferred acidic pH (typical for DyP-type peroxidases), whereas SCDYP2 was most active at pH 8. CONCLUSIONS: Regarding the function of DyPs in nature, these results suggest that availability of different DyP variants with complementary activity profiles in one organism might convey evolutionary benefits. SIGNIFICANCE AND IMPACT OF THE STUDY: DyP-type peroxidases are able to degrade xenobiotic compounds and thus can be applied in biocatalysis and bioremediation. However, the native function of DyPs and the benefits for their producers largely remain to be elucidated.
Subject(s)
Actinobacteria , Peroxidases , Actinobacteria/genetics , Actinobacteria/metabolism , Coloring Agents/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Peroxidases/genetics , Peroxidases/metabolism , Recombinant Proteins/metabolism , Streptomyces , Xenobiotics/metabolismABSTRACT
Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Molecular Chaperones , Animals , Arginine , Chromatography, Liquid , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Halogenation , Hydrophobic and Hydrophilic Interactions , Hypochlorous Acid/chemistry , Imines/metabolism , Lysine , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Proteomics , Tandem Mass SpectrometryABSTRACT
The energy transfer (ET) from carotenoids (Cars) to chlorophylls (Chls) in photosynthetic complexes occurs with almost unitary efficiency thanks to the synergistic action of multiple finely tuned channels whose photophysics and dynamics are not fully elucidated yet. We investigated the energy flow from the Car peridinin (Per) to Chl a in the peridinin chlorophyll a protein (PCP) from marine algae Amphidinium carterae by using two-dimensional electronic spectroscopy (2DES) with a 10 fs temporal resolution. Recently debated hypotheses regarding the S2-to-S1 relaxation of the Car via a conical intersection and the involvement of possible intermediate states in the ET were examined. The comparison with an N89L mutant carrying the Per donor in a lower-polarity environment helped us unveil relevant details on the mechanisms through which excitation was transferred: the ET yield was conserved even when a mutation perturbed the optimization of the system thanks to the coexistence of multiple channels exploited during the process.
Subject(s)
Chlorophyll , Dinoflagellida , Carotenoids/metabolism , Chlorophyll/metabolism , Chlorophyll A/metabolism , Dinoflagellida/metabolism , Energy Transfer , MutationABSTRACT
Cryptophytes are among the few eukaryotes employing phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to cyanobacterial PBP that are organized in membrane-associated phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin 545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. The assembly of PBPs in cryptophytes involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of the eukaryotic S-type PBP lyase GtCPES. We show GtCPES-mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP ß-subunit (PmCpeB) of Prochlorococcus marinus MED4. On the basis of the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB), most likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colored complex in vitro and produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on these findings, we postulate that this single amino acid residue has a crucial role for bilin binding specificity of S-type phycoerythrin lyases but additional factors regulate handover to the target protein.
Subject(s)
Phycobiliproteins , Lyases , Substrate SpecificityABSTRACT
The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with Km values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and Vmax values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg-1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg-1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.
Subject(s)
Actinobacteria/enzymology , Actinobacteria/metabolism , Glutathione Transferase/metabolism , Glutathione/metabolism , Styrenes/metabolism , Actinobacteria/classification , Actinobacteria/genetics , Biotransformation , Epoxy Compounds , Escherichia coli/genetics , Glutathione Transferase/genetics , Ibuprofen , Phylogeny , PlasmidsABSTRACT
[FeFe]-hydrogenases are efficient H2-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic Hox-state and the inactive but oxygen-resistant Hinact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O2-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.
Subject(s)
Crystallography, X-Ray/methods , Binding Sites , Catalytic Domain , Clostridium beijerinckii/enzymology , Clostridium beijerinckii/pathogenicity , Electrochemistry , Kinetics , Models, Theoretical , Spectroscopy, Fourier Transform InfraredABSTRACT
Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.
ABSTRACT
Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.
Subject(s)
Amino Acid Transport Systems, Basic/chemistry , Bacterial Proteins/chemistry , Biosensing Techniques/methods , Ligands , Metabolic Engineering/methods , Amino Acid Transport Systems, Basic/metabolism , Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , Crystallography , Flow Cytometry/methods , High-Throughput Screening Assays/methods , Lysine/metabolism , Microfluidic Analytical Techniques , Models, Molecular , Protein Conformation , Protein Domains , ThermodynamicsABSTRACT
As paradigms for proton-coupled electron transfer in enzymes and benchmarks for a fully renewable H2 technology, [FeFe]-hydrogenases behave as highly reversible electrocatalysts when immobilized on an electrode, operating in both catalytic directions with minimal overpotential requirement. Using the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1) we have conducted site-directed mutagenesis and protein film electrochemistry to determine how efficient catalysis depends on the long-range coupling of electron and proton transfer steps. Importantly, the electron and proton transfer pathways in [FeFe]-hydrogenases are well separated from each other in space. Variants with conservative substitutions (glutamate to aspartate) in either of two positions in the proton-transfer pathway retain significant activity and reveal the consequences of slowing down proton transfer for both catalytic directions over a wide range of pH and potential values. Proton reduction in the variants is impaired mainly by limiting the turnover rate, which drops sharply as the pH is raised, showing that proton capture from bulk solvent becomes critical. In contrast, hydrogen oxidation is affected in two ways: by limiting the turnover rate and by a large overpotential requirement that increases as the pH is raised, consistent with the accumulation of a reduced and protonated intermediate. A unique observation having fundamental significance is made under conditions where the variants still retain sufficient catalytic activity in both directions: An inflection appears as the catalytic current switches direction at the 2H+/H2 thermodynamic potential, clearly signaling a departure from electrocatalytic reversibility as electron and proton transfers begin to be decoupled.
Subject(s)
Hydrogenase/metabolism , Iron-Sulfur Proteins/metabolism , Chlamydomonas reinhardtii , Clostridium , Electron Transport , Hydrogenase/genetics , Iron-Sulfur Proteins/genetics , Mutagenesis, Site-Directed , ProtonsABSTRACT
Hemoglobins (Hbs) utilize heme b as a cofactor and are found in all kingdoms of life. The current knowledge reveals an enormous variability of Hb primary sequences, resulting in topological, biochemical and physiological individuality. As Hbs appear to modulate their reactivities through specific combinations of structural features, predicting the characteristics of a given Hb is still hardly possible. The unicellular green alga Chlamydomonas reinhardtii contains 12 genes encoding diverse Hbs of the truncated lineage, several of which possess extended N- or C-termini of unknown function. Studies on some of the Chlamydomonas Hbs revealed yet unpredictable structural and biochemical variations, which, along with a different expression of their genes, suggest diverse physiological roles. Chlamydomonas thus represents a promising system to analyze the diversification of Hb structure, biochemistry and physiology. Here, we report the crystal structure, resolved to 1.75 Å, of the heme-binding domain of cyanomet THB11 (Cre16.g662750), one of the pentacoordinate algal Hbs, which offer a free Fe-coordination site in the reduced state. The overall fold of THB11 is conserved, but individual features such as a kink in helix E, a tilted heme plane and a clustering of methionine residues at a putative tunnel exit appear to be unique. Both N- and C-termini promote the formation of oligomer mixtures, and the absence of the C terminus results in reduced nitrite reduction rates. This work widens the structural and biochemical knowledge on the 2/2Hb family and suggests that the N- and C-terminal extensions of the Chlamydomonas 2/2Hbs modulate their reactivity by intermolecular interactions.
Subject(s)
Chlamydomonas reinhardtii/chemistry , Hemoglobins/chemistry , Hemoglobins/isolation & purification , Models, Molecular , Protein ConformationABSTRACT
The [FeFe]-hydrogenases catalyze the uptake and evolution of hydrogen with unmatched speed at low overpotential. However, oxygen induces the degradation of the unique [6Fe-6S] cofactor within the active site, termed the H-cluster. We used X-ray structural analyses to determine possible modes of irreversible oxygen-driven inactivation. To this end, we exposed crystals of the [FeFe]-hydrogenase CpI from Clostridium pasteurianum to oxygen and quantitatively investigated the effects on the H-cluster structure over several time points using multiple data sets, while correlating it to decreases in enzyme activity. Our results reveal the loss of specific Fe atoms from both the diiron (2FeH) and the [4Fe-4S] subcluster (4FeH) of the H-cluster. Within the 2FeH, the Fe atom more distal to the 4FeH is strikingly more affected than the more proximal Fe atom. The 4FeH interconverts to a [2Fe-2S] cluster in parts of the population of active CpIADT, but not in crystals of the inactive apoCpI initially lacking the 2FeH. We thus propose two parallel processes: dissociation of the distal Fe atom and 4FeH interconversion. Both pathways appear to play major roles in the oxidative damage of [FeFe]-hydrogenases under electron-donor deprived conditions probed by our experimental setup.